Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics, but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states, and therefore how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three new instances of cross-pathway regulation, which we biochemically verified. These included inhibition of pyruvate kinase by citrate, which accumulated and thereby curtailed glycolytic outflow in nitrogen-limited yeast. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes.
We developed systems to rapidly express any yeast gene or to specifically degrade any protein, each with minimal untargeted disturbance of cell physiology. We illustrate applications of these new tools for elucidating the architecture and dynamics of genetic regulatory networks.
A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
Genome-wide gene-expression studies have shown that hundreds of yeast genes are induced or repressed transiently by changes in temperature; many are annotated to stress response on this basis. To obtain a genome-scale assessment of which genes are functionally important for innate and/or acquired thermotolerance, we combined the use of a barcoded pool of ∼4,800 nonessential, prototrophic Saccharomyces cerevisiae deletion strains with Illuminabased deep-sequencing technology. As reported in other recent studies that have used deletion mutants to study stress responses, we observed that gene deletions resulting in the highest thermosensitivity generally are not the same as those transcriptionally induced in response to heat stress. Functional analysis of identified genes revealed that metabolism, cellular signaling, and chromatin regulation play roles in regulating thermotolerance and in acquired thermotolerance. However, for most of the genes identified, the molecular mechanism behind this action remains unclear. In fact, a large fraction of identified genes are annotated as having unknown functions, further underscoring our incomplete understanding of the response to heat shock. We suggest that survival after heat shock depends on a small number of genes that function in assessing the metabolic health of the cell and/or regulate its growth in a changing environment.
Replicative aging of Saccharomyces cerevisiae is an established model system for eukaryotic cellular aging. A limitation in yeast lifespan studies has been the difficulty of separating old cells from young cells in large quantities. We engineered a new platform, the Miniature-chemostat Aging Device (MAD), that enables purification of aged cells at sufficient quantities for genomic and biochemical characterization of aging yeast populations. Using MAD, we measured DNA accessibility and gene expression changes in aging cells. Our data highlight an intimate connection between aging, growth rate, and stress. Stress-independent genes that change with age are highly enriched for targets of the signal recognition particle (SRP). Combining MAD with an improved ATAC-seq method, we find that increasing proteasome activity reduces rDNA instability usually observed in aging cells and, contrary to published findings, provide evidence that global nucleosome occupancy does not change significantly with age.
SignificanceTrehalose is an important molecule for industrial and medical applications. These applications include use as a food additive to increase sweetness and promote freeze-dry preservation. Trehalose is also included in antibody preparations for stabilization during freezing or desiccation. Further, trehalose biosynthesis is required for virulence of fungal pathogens, and, because animal cells do not synthesize trehalose, trehalose biosynthesis is an attractive antifungal target. Despite all of its uses, the direct physiological roles of trehalose remain unclear. Here, we describe the development and characterization of a system in the model yeast Saccharomyces cerevisiae to directly assess the physiological roles of trehalose. We find that many of the roles traditionally ascribed to trehalose are not the result of trehalose accumulation per se.
Diverse thiol-reactive compounds are found to activate the Hsf1-regulated heat shock response in Saccharomyces cerevisiae. The highly conserved cytosolic Hsp70 protein chaperone is shown to act as a sensor for these molecules through a pair of reactive cysteine residues in the nucleotide-binding domain.
Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37°C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C RTR1 (regulator of transcription). The core RNAPII subunits RPB5, RPB7, and RPB9 were isolated as potent high-copy-number suppressors of the rtr1⌬ temperature-sensitive growth phenotype, and deletion of the nonessential subunits RPB4 and RPB9 hypersensitized cells to RTR1 overexpression. Disruption of RTR1 resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently, rtr1⌬ cells are defective in inducible transcription from the GAL1 promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.Transcription of mRNA and most snRNAs in eukaryotic cells is carried out by the RNA polymerase II (RNAPII) enzyme, consisting of 12 protein subunits (Rpb1 to Rpb12). Five of these (Rpb1, Rpb2, Rpb3, Rpb6, and Rpb11) are homologous to counterparts in bacteria, and six others (Rpb4, Rpb5, Rpb7, Rpb9, Rpb10, and Rpb12) bear resemblance to archaeal RNA polymerase subunits (64). Many of these subunits are also represented by highly conserved homologs in the eukaryotic RNAPI and -III enzymes responsible for rRNA and tRNA synthesis, respectively (52). In addition to the core subunits, numerous additional protein cofactors are required for regulated and accurate gene expression, including the Mediator, Elongator, and SAGA complexes (39). Diverse cellular signals such as environmental stressors (heat, oxidative chemical), nutrient conditions, and proliferation state feed into the transcription machinery at multiple steps (44). One regulatory mechanism involves reversible phosphorylation of the carboxyterminal domain (CTD) within RNAPII (51). The CTD contains multiple repeats of a conserved motif (YSPTSPS) subject to hyperphosphorylation by CTD kinases such as Kin28 and Srb10 (26). Phosphorylation of the Rpb1-CTD at serines in the second and fifth positions within the heptad repeat is associated with transition from preinitiation to a transcriptionally active elongation complex (17). With the scores of known signal inputs, multiple layers of regulation, and hundreds of described components, uncharacterized genes that may play important roles in modulating transcription undoubtedly still exist.One facet...
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